Facilitation of Definitive Cancer Diagnosis With Quantitative Molecular Assays of BRAF V600E and TERT Promoter Variants in Patients With Thyroid Nodules

Key Points Question Does quantitative molecular assay of variant allele fraction (VAF) of genomic variants facilitate cancer diagnosis among patients with thyroid nodules? Findings In this diagnostic study of 378 surgically resected thyroid tumors, sensitive VAF molecular assays were established to elucidate interpatient variability and variation extent of BRAF V600E and TERT promoter variants in thyroid tumors. All tumors harboring either of these variants, whether at low or high VAFs, were diagnosed histopathologically as malignant, and high VAFs of either variant alone or different VAF levels for both variants in coexistence were associated with aggressive histopathologic features and intermediate to high risks of recurrence. Meaning Findings of this study suggest that sensitive VAF assays can quantitatively detect BRAF V600E and TERT promoter variants in thyroid tumors and facilitate a definitive diagnosis of cancer among thyroid nodules by elucidating interpatient variability of oncogenic gene variants in tumors.


Quantitative molecular assays of BRAF V600E and TERT promoter variants (C228T and C250T) using droplet dPCR approach
Quantitative molecular assays of BRAF V600E and TERT promoter variants were performed using droplet dPCR on the QX200 AutoDG Droplet Digital PCR System (Bio-Rad Laboratories, ON, CAN) in the Central Scientific Laboratory of Lunenfeld-Tanenbaum Research Institute, Toronto, Canada. Molecular assay of BRAF V600E variant was conducted to quantify copies of target variant and wild-type in a linear response manner and allow for further quantification of the VAF level with BRAFV600E98 Assay (20X) according to the recently established procedure. 2 With the same strategy, molecular assay was developed to detect and quantify VAFs of TERT variations (C228T and C250T) using LNA probe-based dPCR with TERT Variation Assays (20x). Briefly, droplet dPCR assays were performed in a 22-μL reaction mixture containing 11 μL dPCR Supermix for Probes (2×, no dUTP) (Bio-Rad Laboratories), 1.1 μL of TERT Variation Assay (20×), 3.5 μL of Enhanced solution, 3.0 μL of genomic DNA, and 3.4 μL of deionized water. Droplets were generated using the QX200 Droplet Generator (Bio-Rad Laboratories) and subjected to thermal cycling for amplification under conditions: 1 cycle of 95 °C for 10 min, 45 cycles of 94 °C for 30 s and 54 °C for 1 min, 1 cycle of 98 °C for 10 min with a ramp rate of 2°C/s, and hold at 4 °C. Fractional abundance was acquired by the QX200 droplet reader and calculated with QuantaSoft analysis software (Bio-Rad Laboratories). The TERT variant allele and wild-type allele concentration in the final PCR reaction mix, presented as copies per microliter, were calculated from the values of 6-fluorescein amidite (6-FAM)-positive droplets (variants) and hexachloro-fluorescein (HEX)-positive droplets (wild-type) by applying a fluorescence amplitude threshold. The VAF was computed as percentage of the variant positive droplets of the total positive droplets of TERT wild-type and variants. FTC-133 DNA containing the TERT variant (C228T), BACAP DNA containing the TERT wild-type, and blank control (H2O) were included in each test to verify the assay condition and exclude potential contamination.

Validation of TERT promoter variants by Sanger sequencing
The results of dPCR assay of TERT promoter variants (C228T and C250T) was further validated by power read sequencing. Briefly, the target amplicon was amplified using the NEBNext Ultra II Q5® Master Mix (New England Biolabs, MA, USA) by a nested PCR comprised of the first PCR, with forward 5'-CTTCCCACGTGCGCAGCAGGA and reverse 5'-AGTGGATTCGCGGGCACAGA primers, and the second PCR, with forward 5'-CAGCGCTGCCTGAAACTCG and reverse 5'-ACCCGTCCTGCCCCTTCACCTT primers, at an annealing temperature of 60 o C. After purification of PCR products using the Monarch® DNA Gel Extraction Kit (New England Biolabs), Sanger sequencing was carried out on a power read mode of Applied Biosystems 3730 XL DNA Analyzers (Eurofins Genomics, KY, USA). PCR amplicons from BCPAP and FTC-133 DNA as control were included in each sequencing to verify dPCR assay results.

Assessment of sensitivity, specificity, positive predictive value, negative predictive value and odds ratio
The accuracy and reproducibility of dPCR assay of TERT promoter variants was assessed according to the procedure described previously. 2 In this study, dPCR assays were established for detecting the two TERT promoter variations at a mean (SD) limit of detection 0.03 (0.01) copies/μL in a single test, greatly improving the detection efficiency and sensitivity. Logistic regression analysis was performed to assess the diagnostic value of BRAF and TERT variant assays in identifying patients at an intermediate-to-high risk of recurrence. The clinical sensitivity, specificity, and positive predictive values, negative predictive values and odds ratios with 95% CIs of BRAF V600E and TERT promoter variations for tumor malignancy were calculated using standard formulas in accordance with the Standards for Reporting of Diagnostic Accuracy (STARD) reporting guidelines. 3

Hematoxylin and Eosin staining
Hematoxylin and Eosin (H&E) staining was performed using FFPE tissue sections (4 μm) in Mount Sinai Services (Mount Sinai Hospital, ON, CAN). Briefly, after removal of the wax on the tissue with xylene and hydration via several changes of alcohol, the sections were subjected to a nuclear stain with Harris hematoxylin followed by an eosin counterstain. After passing serials of alcohol to remove all traces of water, the slide was rinsed in several baths of xylene which clears the tissue. A glass coverslip was mounted on a thin layer of polystyrene mountant on the tissue. H&E histology slides were scanned by Microscopy Slide Scanner ZEISS Axio Scan.Z1 (Zeiss, Oberkochen, Germany) in the OPTical IMAging (OPTIMA) Facility at Lunenfeld-Tanenbaum Research Institute in Mount Sinai Hospital and all H&E images were acquired via ZEISS ZEN (blue edition).

Lead contact and method availability
Further information and request for the protocol of dPCR assays should be directed to and will be fulfilled by the Lead Contact, Guodong Fu (David.Fu@sinaihealth.ca; gdfu2002@gmail.com). The flow chart described the potentially eligible patient number identified between March 2016 and March 2020, the eligible number with patient's consent, and the number involved in the quantitative molecular assays and analyses in the current study. The numbers of individuals excluded from the study at each stage were shown due to the lack of patient's consent or no specimens available for research under certain conditions (For example, specimen was not obtained when a tumor was too small or invisible as it was reserved for histology in priority). Specimens were also excluded from analysis when tumors were subsequently identified as microcarcinomas.